In the brain, neurons communicate with other neurons or non-neuronal cells mostly by sending or sensing neurotransmitters or neuromodulators. The ability to detect these compounds in the live brain is essential to understanding brain physiology. Furthermore, the development of methods to measure the amount of neurotransmitters and neuromodulators in vivo is critical to study the large number of pathologies associated with abnormal levels of extracellular signaling molecules in the brain.
In vivo monitoring techniques for neurotransmitters or neuromodulators measure changes in the concentration of specific substances in the extracellular compartment of selected brain regions resulting from the activity of neuronal assemblies. The two principal techniques used in neurotransmitter or neuromodulator monitoring in the live brain are microdialysis and voltammetry.
Microdialysis-based methods consist of implanting a cannula, for instance, inside the brain, to collect submicroliter volumes of cerebrospinal fluid at regular intervals (Day et al., 2001). The microdialysis probe has a diameter ranging from 200-500 μm resulting in a volume resolution of at least 1 mm3 (Portas et al., 2000). Compounds in the samples are commonly separated on a High Performance Liquid Chromatography (HPLC) column, or less frequently, using Capillary Electrophoresis (CE) or capillary Liquid Chromatography (LC) columns (Kennedy et al., 2002). The molecules of interest are detected in the flow-through by analytical methods such as gas chromatography, radioenzymatic assay, radioimmunological measurement, fluorometry, electrochemical detection or mass spectrometry.
The main drawback of microdialysis is its temporal resolution, which can be several orders of magnitude lower than the time scale of electrical activity of brain cells. For example, the neurotransmitter acetylcholine (ACh) is presumably released in the cerebral cortex during short periods, e.g., 250 ms (McCormick et al., 1993). However, a typical microdialysis/HPLC system with femtomole detection limit can analyze a sample every 5-30 min (Day et al., 2001). If the microdialysate is analyzed via capillary electrophoresis combined with laser-induced fluorescence, nanoliter samples may be collected and the temporal resolution reduced to ˜60 s (Lena et al., 2005). However, laser-induced fluorescence requires a method to conjugate a fluorophore to the analyte. Further, experimental results using capillary electrophoresis combined with fluorescence for most neurotransmitters, including ACh, have not been published to date. Detection of ACh by tandem mass spectrometry still requires several minutes per sample (Shackman et al., 2007; Zhang et al., 2007).
Temporal resolution of microdialysis systems can be improved at the expense of sensitivity, making detection of neuroactive substances problematic. To circumvent this problem, substances known to increase levels of neurotransmitters are included in the microdialysis perfusate (e.g., Himmelherber et al., 1998). In this respect, the practice of adding acetylcholine esterase inhibitors for ACh measurements is particularly controversial as the inhibitors perturb the physiology by artificially raising levels of ACh in the brain (Day et al., 2001).
Direct in vivo electrochemical methods rely on implanted electrodes measuring the redox current generated by the substances of interest at the electrodes. These techniques, referred to as amperometry, chronoamperometry and fast-scanning voltammetry, differ mostly in the stimulation waveform applied to the measuring electrode (Michael and Wightman, 1999).
Electrochemical methods have been used to detect, for instance, plasma glucose levels. In the brain, the most successful use of electrochemistry has been in the detection of dopamine, with a temporal resolution below one second (Robinson et al., 2003). Although detection of choline looks promising, electrochemical detection of ACh has proven difficult and current designs have a detection limit of 80 nM-660 nM (Mitchell, 2004; Bruno et al., 2006) in vitro, whereas estimates of basal levels using microdialysis are on the order of 4 to 100 nM (e.g. Rasmusson et al., 1992; Jimenez-Capdeville and Dykes, 1996; Himmelheber et al., 1998).
The main drawback of electrochemical methods is the lack of chemical sensitivity (Michael and Wightman, 1999). Electrochemical methods are, for instance, unable to differentiate between norepinephrine and dopamine (Robinson et al., 2003). Selectivity can be enhanced by coating electrodes, but at the expense of temporal resolution. Furthermore, electrochemical measurements are often contaminated by signals from precursors or metabolites of neurotransmitters, or unrelated compounds. For instance serotonin electrodes detect two metabolites of serotonin, 5-hydroxyindolacetic acid and uric acid (Cespuglio et al., 1998; Nakazato and Akiyama, 1999).
The difficulty in measuring single biochemical molecules in-vivo is even greater when multiple molecules need to be specifically detected simultaneously at the same site. Studies using voltammetry demonstrated the possibility to record several substances at the same electrode, but with the same selectivity shortcomings as described above (e.g., Nakazato and Akiyama, 1999). As such, neither microdialysis nor electrochemical techniques are adequate to measure simultaneously and unambiguously two biochemicals.
A recent approach, called FLIPE (Fluorescent indicator protein for glutamate), uses a genetically engineered chimeric protein composed of a glutamate binding site flanked by a blue and a yellow fluorescent protein (Okumoto et al., 2005). Following cell surface expression of the FLIPE sensor, binding of glutamate to the protein elicits a conformational change leading to a Fluorescence Resonance Energy Transfer (FRET) from the blue to the yellow fluorophore, which can be optically detected.
The main drawback of this method is its lack of flexibility, since it entails (1) finding a natural binding protein for the molecule of interest, from which the binding site is derived (2) modifying the chimera to provide enough molecular motion for FRET to occur, a step that requires extensive mutagenesis and screening, and (3) expressing the FLIPE sensor in the cells of interest in the brain.
Accordingly, the need remains for a method for a sensitive and specific in vivo detection of neurotransmitters with good temporal resolution.